Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition

Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d-21, -14, -7,0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LM FC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.     

Ultrasonographic evaluation of urinary bladder in normal,fern fed and enzootic bovine haematuria-affected cattle

A total of 19 adult hill cattle of both sexes were subjected to trans-rectal ultrasound scanning of urinary bladder to evaluate bladder wall thickness and the presence of space-occupying lesions. The animals were divided into four groups. Eight apparently healthy hill cattle maintained under standard ration served as control (group I) and the remaining II animals were divided into three groups (II, III and IV). Group II animals (n = 8) were fed with different type of ferns which were further divided into subgroups II-P, -D and -B and fed with Polystichum squarrosom (n = 2). Dryopteris juxtaposita (n = 2) and Pteridium aquilinum (n = 4) ferns, respectively. The one animal in group III was a natural case of enzootic bovine haematuria (EBH) and the two animals in group IV were natural cases of microscopic EBH fed with Polystichum squarrosum fern. In group I animals, the average bladder wall thickness was 1.45 mm. The delineation of the bladder wall was uniformly smooth and the echo pattern of the bladder was homogeneously black, which was suggestive of clear urine content. In group II (P, D and B) the average bladder wall thickness of the six animals was 1.87 mm and the sonographic features were within normal limit when compared with controls. In two of the animals of group II-B, the bladder wall was apparently thick (4.36 mm) and there was no intraluminal mass except at one or two focal elevated points. Animals of groups III and IV showed the average bladder wall thickness of 4.86 mm and were characterized by the presence of irregular sessile masses extending into the bladder lumen. The homogeneous anechoic area was reduced centrally due to the presence of a hypoechoic soft tissue mass all around the bladder wall. Post-sonographic urinalysis, biopsy and necropsy of selected cases further confirmed the sonographic findings.     

The influence of age and gender on haematological parameters in Lipizzan horses

Haematological parameters [red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), haemoglobin concentration, mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC)] in resting Lipizzan horses were determined for 143 stallions, 104 mares and 25 foals. The mean RBC and WBC values in Lipizzans were in the lower part of the normal range for warm-blooded horses. The mean PCV, MCV and MCH values were higher, but the mean haemoglobin concentration and MCHC values were lower than reported for other warm-blooded horses. In foals, the mean RBC, WBC, PCV, haemoglobin concentration and MCHC values were higher, whereas MCV and MCH were lower than in older animals. Results indicating a significant decrease in WBC (P < 0.01) and an increase in MCV, MCH and MCHC (P < 0.05) with increasing age are consistent with some other reports on warm-blooded horses. The age-related variations in RBC and PCV were less marked. Contrary to some reports, RBC (P < 0.01), WBC and haemoglobin concentration (P < 0.001) were higher in Lipizzan stallions than in mares, but differences in MCHC, MCH and MCV were insignificant. The specific haematological values determined in Lipizzans are presumably a result of selection and should be taken into consideration when dealing with this race of horses.     

Susceptibility of trypanotolerant West African Dwarf goats and F1 crosses with the susceptible Sahelian breed to experimental Trypanosoma congolense infection and interactions with helminth infections and different levels of diet

Forty pure West African Dwarf (WAD) goats and 35 of its F1 crosses with the Sahelian breed were used in a multifactorial experimental design to evaluate the effects of an experimental Trypanosoma congolense infection and interactions with natural helminth infections and different levels of diet on health and productivity of these two breeds. Trypanosome infection caused a severe drop in packed cell volume (PCV), but this was not significantly affected by breed. Neither deworming nor diet had any effect on the course of anaemia after trypanosome infection. The mean score of parasitaemia tended to be higher in crossbreeds than in WAD goats although this was not significant (P>0.05). Similarly, the antibody response to trypanosome infection was not significantly different between breeds. Parasitaemia level was significantly influenced by the level of diet with the group under high supplementation having a higher mean parasitaemia score than the group under low supplementation. Weight loss due to trypanosome infection tended to be greater in crossbreeds than in WAD goats (P>0.05).During this study, there was no difference in mean helminth egg output between crossbred and WAD goats. However, between weeks 4 and 10 after trypanosome infection (corresponding to a period of heavy rainfall and highly infective pastures), the mean egg output was higher in the crossbreeds.The immunosuppressive effect of trypanosome infections was revealed by a lower antibody response to Haemonchus contortus in infected animals compared to the uninfected controls. Trypanosome infection tended to increase strongyle egg output. This study did no reveal any superior trypanotolerance of WAD goats compared to crossbreeds.     

A cross-sectional coprological survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines

A cross-sectional coprological survey of liver flukes (Fasciola hepatica and Dicrocoelium dendriticum) was conducted on 81 bovine farms and 197 ovine farms with animals pasturing in an area (3971 km(2)) of the southern Italian Apennines. The farms were selected to be uniformly distributed throughout the study area using geographical information system (GIS) software. Between June 1999 and March 2000, faecal samples were collected from 975 cattle and 3940 sheep and examined using a modified McMaster technique. The results were subjected to statistical analysis and point distribution maps (PDMs) were drawn by GIS.Cattle of 9 of the 81 (11.1%) farms were positive for F. hepatica and of 43 (53.1%) for D. dendriticum. Sheep of 8 of the 197 (4.1%) farms were positive for F. hepatica and of 133 (67.5%) for D. dendriticum. Co-infection was found in cattle of 2 (2.5%) farms, and in sheep of 8 (4.1%) farms.The findings of the present survey show that D. dendriticum was the predominant liver fluke found in cattle and sheep with respect to egg count numbers for both farms and animals. In addition, the general trends of the PDMs show that D. dendriticum was widely and homogeneously spread throughout the study area, whereas F. hepatica was present only in a few concentrated zones of the study area that had both positive bovine and positive ovine farms.     

Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples

The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.     

Effects of cross-tying horses during 24 h of road transport

Transportation stress has been implicated as a predisposing factor to respiratory disease in horses. Cross-tying horses individually in stalls is common practice for transporting show and racehorses, but horses also travel in small groups or individually without being restricted by tying. The objective of this study was to compare physiological responses of horses travelling cross-tied or loose during 24 h of road transport. Ten horses were used in a cross-over design consisting of two 4 day trials. In the first trial, 6 horses were cross-tied, while 2 pairs of horses were loose in enclosed compartments. Treatments were reversed in the second trial. Baseline samples were collected on Day 1, horses transported on Day 2, and recovery data collected on Days 3 and 4. Blood samples were collected daily at 0800, 1100 and 2000 h. The mean responses in all horses of serum cortisol, lactate, glucose, alpha1-acid glycoprotein, and total protein concentrations, packed cell volume (PCV), white blood cell (WBC) counts and aminotransferase and creatine kinase were was elevated significantly from baseline during the 4 day study. The response of white blood cell counts, neutrophil to lymphocyte ratios and glucose and cortisol concentrations was significantly elevated in the cross-tied compared to the loose group during transport and recovery. This study supports the recommendation of allowing horses during long-term transportation to travel loose in small compartments, without elevating their head by cross-tying.     

Plasma adrenocorticotropin (ACTH) concentrations and clinical response in horses treated for equine Cushing's disease with cyproheptadine or pergolide

Plasma ACTH levels have been variable in horses with a positive clinical response for therapy for equine Cushing's Disease (ECD). Therefore, our purpose was to determine the value of monitoring plasma adrenocorticotropin (ACTH) levels during treatment of equine Cushing's disease (ECD) with either cyproheptadine (n = 32) or pergolide (n = 10). First, we validated the chemiluminescent ACTH assay (specificity, precision, accuracy, intra-assay and interassay variations) and tested methods of handling the whole blood from the time of collection to when the ACTH was assayed. The sensitivity and specificity of high plasma ACTH levels for detecting ECD was determined in a retrospective study on hospitalised horses (n = 68). Surveys were sent to veterinarians who submitted equine ACTH levels that were high initially and had at least 2 ACTH samples to determine the value of monitoring ACTH levels during therapy of ECD. The ACTH chemiluminescent assay was valid. The ACTH was stable when whole blood was collected and held in plastic tubes for 8 h before separating the plasma. The sensitivity and specificity of plasma ACTH levels for detecting ECD were 84% (n = 19,95% CI 60,97) and 78% (n = 49,95% CI 63,88), respectively. Treated horses generally showed a decrease in plasma ACTH. Plasma ACTH levels may be helpful when monitoring therapy of ECD, although improvement in clinical signs should be considered most important. There were no differences between cyproheptadine and pergolide in terms of improvements in any of the clinical signs.     

Effect of different fat sources on in vitro degradation of nutrients and certain blood parameters in sheep

This study was designed to determine the effects of calcium salt of palm oil fatty acids (CS), hydroxyethylsoyamide (HESA), butylsoyamide (BSA) and soybean oil (SO) on degradation of crude protein and fibre in vitro, and on the blood plasma lipid parameters in vivo. Five mature wethers (body weight 75 kg) were fed five diets in a 5 x 5 Latin square experiment. The control diet consisted of 50% meadow hay and 50% concentrate with no added fat. The control diet was supplemented with CS, HESA, BSA, or SO. Fat was added at 3.5% of dietary dry matter (DM). The final ether extract content of the ration was near 6%. Each period lasted 20 days. Fat supplements, except HESA, consistently decreased the in vitro DM disappearance of soybean meal as compared to control. In contrast to the effect of other treatments, crude protein degradation was greatest in the test tubes with inocula obtained from sheep fed diet with HESA. Fat supplements equally inhibited the DM and fibre breakdown of alfalfa pellet. CS and HESA seemed to be less detrimental to in vitro fermentation of neutral detergent fibre (NDF) than BSA and SO. All fat supplements increased blood plasma triglyceride, cholesterol and total lipid content. Plasma concentration of cholesterol and total lipid was highest with SO. The inclusion of CS in the diet increased 16:0, while all fat supplements increased plasma 18:0 and decreased 16:1 and 18:1 fatty acid content. Plasma 18:2n-6 was not changed by feeding CS and SO. However, compared to the control diet, 18:2n-6 increased with 12 and 41% in plasma fatty acids when sheep were fed HESA and BSA, respectively. The results showed that plasma concentration of linoleic acid was enhanced more when the amide was synthesised from butylamine than when from ethanolamine.     

Identification of immunoreactive polypeptides of Babesia equi parasite during immunization

This study was carried out to identify immunoreactive polypeptides in Babesia equi merozoite antigen. Three fractions of killed B. equi merozoite antigen viz.; whole merozoite (WM), cell membrane (CM) and high speed supernatant (HSS) antigens were prepared from the parasite infected erythrocytes. These antigenic preparations along with ghost antigen from non-infected erythrocytes were fractionated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with sera showing high antibody titres. On SDS-PAGE, 16 polypeptides with molecular weight (Mr) in the range of 112-17kDa were obtained from the WM and CM antigens. But only six polypeptides were detected (96.5-28kDa) in the HSS antigen. On immunoblotting with high titred serum collected from donkeys following two immunizations with a killed B. equi merozoite immunogen, 11 polypeptides were observed in the WM and CM antigens (Mr 112-18kDa). Of these, four polypeptides (Mr 112, 45, 33 and 18kDa) were identified as most immunoreactive. Besides these, a 28kDa was observed as strong immunoreactive protein in WM and CM antigens. The HSS antigen showed only six polypeptides and one peptide (28kDa) was identified as immunoreactive. When high titred serum collected from immunized donkeys following challenge with B. equi infected blood and was used for immunoblotting, the protein profile of WM and CM antigens remained the same. However, three additional polypeptides (Mr 81, 54.5 and 39kDa) were detected in HSS antigen.